Highly specific and sensitive radioimmunoassays coupled to HPLC and gel filtration techniques were utilized to identify secretin, motilin and pancreatic polypeptide immunoreactivity in the brain of the rat. Immunocytochemical studies revealed a wide distribution of pancreatic polypeptide (PP) immunoreactive cell bodies and nerve fibers in the rat central and peripheral nervous systems. A number of PP-immunoreactive cells were demonstrated to coexist with catecholamine neurons in the pons-medulla and sympathetic ganglia. Motilin-like immunoreactive nerve fibers and cell bodies were observed in the hypothalamus, preoptic areas and Purkinje cells of the cerebellum. Radioimmunological studies revealed a wide distribution pattern of secretin in the brain of the rat. A brain-pituitary pathway for secretin has also been identified. Intraventricular injection of secretin reduced open field activity and the number of novel-object approaches in rats. Secretin also decreased respiration rate in anesthetized rats and increased defecation in awake rats. Intravenous injection of secretin in anesthetized hydrated rats caused a dose-related antiduretic effert.